Biodegradation of the Steroid Progesterone in Surface Waters A Thesis Submitted for the Degree of Doctor of Philosophy By Jasper Oreva Ojoghoro Institute of the Environment, Health and Societies May, 2017 Abstract Many studies measuring the occurrence of pharmaceuticals, understanding their environmental fate and the risk they pose to surface water resources have been published. However, very little is known about the relevant transformation products which result from the wide range of biotic and abiotic degradation processes that these compounds undergo in sewers, storage tanks, during engineered treatment and in the environment. Thus, the present study primarily investigated the degradation of the steroid progesterone (P4) in natural systems (rivers), with a focus on the identification and characterisation of transformation products. Initial work focussed on assessing the removal of selected compounds (Diclofenac, Fluoxetine, Propranolol and P4) from reed beds, with identification of transformation products in a field site being attempted. However, it was determined that concentrations of parent compounds and products would be too low to work with in the field, and a laboratory study was designed which focussed on P4. Focus on P4 was based on literature evidence of its rapid biodegradability relative to the other model compounds and its usage patterns globally. River water sampling for the laboratory-based degradation study was carried out at 1 km downstream of four south east England sewage works (Blackbirds, Chesham, High Wycombe and Maple Lodge) effluent discharge points. Suspected P4 transformation products were initially identified from predictions by the EAWAG Biocatalysis Biodegradation Database (EAWAG BBD) and from a literature review. At a later stage of the present work, a replacement model for EAWAG BBD (enviPath) which became available, was used to predict P4 degradation and results were compared. Samples were analysed using low resolution and accurate-mass time-of-flight mass spectrometers. Three degradation studies were conducted. Sampling for all studies was carried out at the same time in the year to minimize temporal variability in conditions and allow for effective comparison of results. Androgenic and progesterone yeast screens were carried out to assess the biological activity of transformation products. i Overall, the present study indicates that P4 degradation is relatively rapid in surface waters. Degradation followed the first order degradation kinetic and spiked P4 concentration (1,000 µg/L) was degraded below instrument detection limit (0.32 µg/L) in 72 h (3 days) in all locations except High Wycombe where 90% degradation occurred. Degradation half-life (t½) was approximately 7 hours in all sites except High Wycombe (29 hours) where some residual P4 (100 μg/L) was present after 72 hours. Overall, degradation rate was faster (0.1 hours) in the present study than those reported for algae (0.02-0.04 hours), thus, indicating that a more diverse microbial community was responsible for the breakdown of P4 observed in the present study. A number of putative degradation products were identified, including boldenone and (with less certainty) testosterone. The results highlighted that the transformation of progesterone can potentially create other classes of steroids, some of which may still be potent, and possess other types of biological activity. Although progesterone degraded to an androgen (boldenone) as shown by chemical analysis, the concentrations in the environment are unlikely to represent any immediate threat to aquatic resources, as results of the androgen screen did not show any androgenic activity when working at elevated (1,000 μg/L) initial progesterone concentrations. ii Declaration I hereby declare that the work described in this thesis were carried out independently by the author between 2014 and 2017 and has not been submitted anywhere for any award. Where other sources of information have been used, they have been appropriately referenced and all support received have been duly acknowledged. Jasper Oreva OJOGHORO May, 2017 iii Dedication This work is dedicated to Jehovah God for His abundant mercies, wisdom and grace upon me throughout my research. Also, to my lovely wife Mrs Victoria Oghenekaro Jasper-Ojoghoro and my adorable son Master Jason Oghenekome Jasper-Ojoghoro for love, devotion, support and encouragement. iv Acknowledgement I am profoundly grateful to the Nigeria Tertiary Education Trust Fund (TETFund) for providing the funding for this study. I am also grateful to the Delta State University, Abraka for facilitating my funding application and for granting my study leave application to complete the present study. I am also grateful to the Uduaghan led-administration of the Delta State Government and the 2010- 2015 board members of the Delta State Scholarship Board for providing the starting funds for this study. I am very grateful to my supervisor, Dr Mark D. Scrimshaw for going above and beyond in supervising me. His guidance, support, patience and encouragement, contributed immensely to the success of this study. I am also very grateful to Dr Chaudhary Abdul, my second supervisor for always giving the needed incite and perspective into my research. I am hugely indebted to Professor John Sumpter, who though very busy and not a member of my supervisory team, played a huge advisory role in my research. I am grateful to Dr Pablo Campo Moreno, Nicola Beresford, Julie Walker, Neil Brodigan, Amy Ferreira, Giles Drinkwater, Marta Straszkiewicz, Dr Belinda Huerta-Buitrago and Dr Alice Baynes for their technical support. I am also grateful to other staff in Environmental Sciences and my fellow PhD students who supported me at one point or the other. I am grateful to Professor (Mrs) M. Agbogidi, Professor (Mrs) N. Edema, Professor V. Peretomode, Professor S. Asagba, Professor H. Eghagha, Professor Atubi, Professor Anigala, Professor T. Akporhunor, Professor and Mrs O. Anomohanran, Professor C. Orubu, Dr C. Onogbosele, Mr and Mrs S. Joseph, Lt. K. Renner, Mr Sam Okolo, Mr C. Okwuokei, Mr and Mrs E. Uduboh, Mr and Mrs Odiete, Mr Afoke Evovo, Mr and Mrs A. Agbrara, Mr and Mrs G. Ogboru, Mr and Mrs A. Onwubiko, Mr and Mrs A. Adekanmi, Mr and Mrs V. Ajayi, Mr and Mrs B. Eradajire, Miss M. Denedo, Mrs O. Akpobi and Mrs G. Obi-Iyeke for their invaluable support. I am most dearly appreciative of my lovely wife Mrs Victoria Oghenekaro Jasper-Ojoghoro and my adorable son Master Jason Oghenekome Jasper-Ojoghoro for love, devotion, support and encouragement. My parents Mr and Mrs Vincent Ojoghoro, Mr and Mrs Collins Ogoru and siblings are also not left out for their prayerful support. v Table of Content Abstract ................................................................................................................................. i Declaration............................................................................................................................ iii Dedication ............................................................................................................................. iv Acknowledgement ................................................................................................................. v Table of Content ................................................................................................................... vi List of Figures ....................................................................................................................... x List of Tables ..................................................................................................................... xvii List of Abbreviations ............................................................................................................. xx List of Publications ............................................................................................................. xxv List of Equations ............................................................................................................... xxvi Chapter One: Introduction ..................................................................................................... 1 1.0 Introduction ............................................................................................................. 2 1.1 Sources of Pharmaceuticals in the Water Environment ........................................... 5 1.2 Research Aim and Objectives ............................................................................... 10 Chapter Two: Literature Review .......................................................................................... 12 2.0 Introduction ........................................................................................................... 13 2.1 Transformation of Pharmaceuticals ....................................................................... 16 2.1.1 The Potency of Natural and Synthetic Steroids and the Effects of Structural Transformation ............................................................................................................. 26 2.1.2 Role of TPs in Observed but Unexplained Biological Activity ......................... 28 2.1.3 Key Transformation Pathways in the Environment ......................................... 30 2.2 Approaches to Studying Degradation and Identification of TPs ............................. 40 2.2.1 Analytical Procedure for the Detection and Identification of TPs .................... 42 vi 2.3 Biotransformation Prediction